α/β-Hydrolase domain-containing 6 (ABHD6) accelerates the desensitization and deactivation of TARP γ-2-containing AMPA receptors
- School of Basic Medical Sciences, Beijing Key Laboratory of Neural Regeneration and Repair, Advanced Innovation Center for Human Brain Protection, Capital Medical University, China
- Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, China
- School of Basic Medical Sciences, Wenzhou Medical University, China
- Ministry of Education Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Department of Neurology, Nanjing Drum Tower Hospital, Medical School, Nanjing University, China
- Chinese Institute for Brain Research, China
- State Key Laboratory of Translational Medicine and Innovative Drug Development, Jiangsu Simcere Pharmaceutical Co. Ltd, China
Figures
Figure 1 with 1 supplement
The effect of overexpression of ABHD6 on the reduction of peak current in AMPARs.
(A) Plasmid abbreviations and variant combinations of AMPAR. (B–D) Representative traces (left) and summary graphs of the peak amplitudes (right) of 10 mM glutamate-induced currents in HEK 293T cells transfected with GluA1–3 (black), GluA1−3 + ABHD6 (orange), GluA1−3 + TARP γ-2 (blue), and GluA1−3 + TARP γ-2 + ABHD6 (red). The statistical method was one-way ANOVA followed by a two-way comparison (*p < 0.05; **p < 0.01; ***p < 0.001; Supplementary file 2).
Figure 1—figure supplement 1
Schematic illustration of the AMPAR subunit; the sequence alignment of RNA splice variants and editing of AMPAR; co-expression validation and Pearson’s correlation between natural logarithm peak amplitude (pA) and the τw, deact and τw, des co-expression with various GluA subunits.
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Figure 1—figure supplement 1—source data 1
The raw, uncropped blot images of Figure 1—figure supplement 1 D1-2.
- https://cdn.elifesciences.org/articles/99623/elife-99623-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
The uncropped blots with the relevant bands clearly labelled of Figure 1—figure supplement 1 D1-2.
- https://cdn.elifesciences.org/articles/99623/elife-99623-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
Overexpression of ABHD6 accelerated the deactivation of AMPARs–TARP γ-2 complexes in HEK 293T cells.
The normalized traces and the summary bar graphs of the τw, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch from HEK 293T cells transfected with GluA (black), GluA + ABHD6 (orange), GluA +TARP γ-2 (blue), and GluA + TARP γ-2 + ABHD6 (red). (A) GluA1i, (B) GluA1o, (C) GluA2(Q)i-R, (D) GluA2(Q)o-R, (E) GluA2(Q)i-G, (F) GluA2(Q)o-G, (G) GluA2(Q)i-R–TARP γ-2 tandem, and (I) GluA1i–TARP γ-2 tandem. γ2-containing GluA receptors could be isolated when 50 μM spermine was in the internal solution and recorded at +50 mV, the average traces and the normalized traces (right), and the summary bar graphs of the τw, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at +50 mV from HEK 293T cells transfected with GluA–TARP γ-2 tandem (blue) and GluA–TARP γ-2 tandem + ABHD6 (red). (H) GluA2(Q)i-R–TARP γ-2. (J) GluA1i–TARP γ-2. The statistical method was one-way ANOVA followed by a two-way comparison (*p < 0.05; **p < 0.01; ***p < 0.001; Supplementary file 4).
Figure 2—figure supplement 1
Average traces of deactivation of AMPAR with overexpression of ABHD6 in HEK 293T cells.
The average traces of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch from HEK 293T cells transfected with GluA (black), GluA + ABHD6 (orange), GluA + TARP γ-2 (blue), and GluA + TARP γ-2 + ABHD6 (red). (A) GluA1i, (B) GluA1o, (C) GluA2(Q)i-R, (D) GluA2(Q)o-R, (E) GluA2(Q)i-G, (F) GluA2(Q)o-G, (G, H) GluA2(Q)i-R–TARP γ-2 tandem. (I, J) GluA1i–TARP γ-2 tandem.
Figure 3 with 1 supplement
Overexpression of ABHD6 accelerated the desensitization of AMPARs–TARP γ-2 complexes in HEK 293T cells.
The normalized traces and the summary bar graphs of the τw, des of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch from HEK 293T cells transfected with GluA (black), GluA + ABHD6 (orange), GluA + TARP γ-2 (blue), and GluA + TARP γ-2 + ABHD6 (red). (A) GluA1i, (B) GluA1o, (C) GluA2(Q)i-R, (D) GluA2(Q)o-R, (E) GluA2(Q)i-G, (F) GluA2(Q)o-G, (G) GluA2(Q)i-R–TARP γ-2 tandem, and (I) GluA1i–TARP γ-2 tandem. TARP γ-2-containing GluA receptors could be isolated when 50 μM spermine was in the internal solution and recorded at +50 mV, the average traces and the normalized traces (right), and the summary bar graphs of the τw, des, and peak amplitude of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at +50 mV from HEK 293T cells transfected with GluA–TARP γ-2 tandem (blue) and GluA–TARP γ-2 tandem + ABHD6 (red). (H) GluA2(Q)i-R–TARP γ-2. (J) GluA1i–TARP γ-2. The statistical method was one-way ANOVA followed by a two-way comparison (*p < 0.05; **p < 0.01; ***p < 0.001; Supplementary file 6).
Figure 3—figure supplement 1
Average traces of desensitization of AMPAR with overexpression of ABHD6 in HEK 293T cells.
The average traces of glutamate (10 mM Glu, 500 ms) induced currents in the outside-out patch from HEK 293T cells transfected with GluA (black), GluA + ABHD6 (orange), GluA + TARP γ-2 (blue), and GluA + TARP γ-2 + ABHD6 (red). (A) GluA1i, (B) GluA1o, (C) GluA2(Q)i-R, (D) GluA2(Q)o-R, (E) GluA2(Q)i-G, (F) GluA2(Q)o-G, (G, H) GluA2(Q)i-R–TARP γ-2 tandem, and (I, J) GluA1i–TARP γ-2 tandem.
Figure 4 with 1 supplement
Overexpression of ABHD6 slows the recovery from desensitization of GluA1i–TARP γ-2 complexes in HEK 293T cells.
(A–F) Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA (black), GluA + ABHD6 (orange), GluA + TARP γ-2 (blue), and GluA + TARP γ-2 + ABHD6 (red). The recovery ratio curves from desensitization and the summary bar graphs of the τw, rec. (A) GluA1i, (B) GluA1o, (C) GluA2(Q)i-R, (D) GluA2(Q)o-R, (E) GluA2(Q)i-G, (F) GluA2(Q)o-G, (G) GluA2(Q)i-R–TARP γ-2 tandem, and (H) GluA1i–TARP γ-2 tandem. The statistical method was one-way ANOVA followed by a two-way comparison (*p < 0.05; **p < 0.01; ***p < 0.001; Supplementary file 8).
Figure 4—figure supplement 1
Typical traces of the recovery from desensitization of AMPAR in HEK 293T cells.
(A–F) Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA (black), GluA + ABHD6 (orange), GluA + TARP γ-2 (blue), and GluA + TARP γ-2 + ABHD6 (red). The first application of 100 ms glutamate was followed by a second glutamate application at increasing pulse intervals at –60 mV. The typical traces from a cell are normalized and aligned to the peak. The typical traces of the recovery from desensitization. (A) GluA1i, (B) GluA1o, (C) GluA2(Q)i-R, (D) GluA2(Q)o-R, (E) GluA2(Q)i-G, (F) GluA2(Q)o-G, (G) GluA2(Q)i-R–TARP γ-2 tandem, and (H) GluA1i–TARP γ-2 tandem.
Figure 5 with 1 supplement
Overexpression of ABHD6 accelerated the deactivation and desensitization of GluA1i/GluA2(R)i-G (or GluA2(R)i-G/GluA3(R)i) receptors–TARP γ-2 complexes in HEK 293T cells and slowed the recovery of GluA1i/GluA2(R)i-G receptors in the presence and absence of TARP γ-2.
(A) The normalized traces and the summary bar graphs of the τw, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at –60 mV from HEK 293T cells transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 + ABHD6 (red). (B) The normalized traces and the summary bar graphs of the τw, des, and peak amplitude of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at –60 mV from HEK 293T cells transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 + ABHD6 (red). (C, D) Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2+ABHD6 (red). The first application of 100 ms glutamate was followed by a second glutamate application at increasing pulse intervals at –60 mV. The recovery ratio curves from desensitization (C) and the summary bar graphs of the τw, rec (D). The statistical method was one-way ANOVA followed by a two-way comparison (*p < 0.05; **p < 0.01; ***p < 0.001; Supplementary file 10 and Supplementary file 12).
Figure 5—figure supplement 1
Average traces of the deactivation, desensitization, and recovery from desensitization of GluA1i/GluA2(R)i-G (or GluA2(R)i-G/GluA3(R)i) receptors–TARP γ-2 complexes in HEK 293T cells.
(A) The average traces of the τw, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at –60 mV from HEK 293T cells transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 + ABHD6 (red). (B) The average traces of the τw, des, and peak amplitude of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at –60 mV from HEK 293T cells transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 + ABHD6 (red). (C) The typical trace of recovery from desensitization. Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i (black), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + ABHD6 (orange), GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 (blue), and GluA1i/GluA2(R)i-G or GluA2(R)i-G/GluA3(R)i + TARP γ-2 + ABHD6 (red). The first application of 100 ms glutamate was followed by a second glutamate application at increasing pulse intervals at –60 mV.
Figure 6
Deletion of ABHD6 decelerated the deactivation and desensitization in ABHD6 KO primary hippocampus neurons.
(A) The normalized traces and the summary bar graphs of the τw, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at –70 mV from wild-type primary hippocampus neurons (black) and ABHD6-knockout (ABHD6 KO) primary hippocampus neurons (red); (A2) the average traces of the τw, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at –70 mV from primary hippocampus neurons. (B) The normalized traces and the summary bar graphs of the τw, des of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at –70 mV from wild-type primary hippocampus neurons (black) and ABHD6-knockout (ABHD6 KO) primary hippocampus neurons (red); (B2) the average traces of the τw, des of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at –70 mV from primary hippocampus neurons. (C) Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from wild-type primary hippocampus neurons (black) and ABHD6-knockout (ABHD6 KO) primary hippocampus neurons (red). The first application of 100 ms glutamate was followed by a second glutamate application at increasing pulse intervals at –70 mV. The recovery ratio curves from desensitization (C1, left), the summary bar graphs of the τw, rec (C2, right), and the typical trace of recovery from desensitization (C2). (D) The typical trace and the summary bar graphs of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch from WT primary hippocampus neurons (black) and ABHD6-knockout (ABHD6 KO) primary hippocampus neurons (red). The statistical method was one-way ANOVA followed by a two-way comparison (*p < 0.05; **p < 0.01; ***p < 0.001; Supplementary file 14).
Figure 7 with 1 supplement
ABHD6 accelerated the deactivation and desensitization of homomeric GluA4i–TARP γ-2 complexes and negatively regulates the kinetics of GluA1i–TARP γ-8 complexes.
(A) The normalized traces and the summary bar graphs of the τw, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at –60 mV from HEK 293T cells transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i + TARP γ-2 + ABHD6 (red). (B) The normalized traces and the summary bar graphs of the τw, des of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at –60 mV from HEK 293T cells transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i + TARP γ-2 + ABHD6 (red). (C, D) Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i + TARP γ-2 + ABHD6 (red). The statistical method was one-way ANOVA followed by a two-way comparison (*p < 0.05; **p < 0.01; ***p < 0.001; Supplementary file 16, Supplementary file 18).
Figure 7—figure supplement 1
Average traces of the deactivation, desensitization, and recovery from desensitization of GluA4i–TARP γ-2 complexes and GluA1i–TARP γ-8 complexes in HEK 293T cells.
(A) The average traces of the τw, deact of glutamate (10 mM Glu, 1 ms) induced currents in the outside-out patch recorded at –60 mV from HEK 293T cells transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i +TARP γ-2 + ABHD6 (red). (B) The average traces of the τw, des, and peak amplitude of glutamate (10 mM Glu, 100 ms) induced currents in the outside-out patch recorded at –60 mV from HEK 293T cells transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i + TARP γ-2 + ABHD6 (red). (C) The typical trace of recovery from desensitization. Glutamate (Glu, 10 mM) induced currents in an outside-out patch excised from an HEK 293T cell transfected with GluA4i or GluA1i (black), GluA4i or GluA1i (black) + ABHD6 (orange), GluA4i or GluA1i + TARP γ-2 (blue), or GluA4i or GluA1i + TARP γ-2 + ABHD6 (red). The first application of 100 ms glutamate was followed by a second glutamate application at increasing pulse intervals at –60 mV.
Additional files
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Supplementary file 1
Summary of peak amplitude (pA) of GluAs when co-transfected with/without γ-2 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp1-v1.docx
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Supplementary file 2
Summary of p values for comparison of peak amplitude.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp2-v1.docx
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Supplementary file 3
Summary of τw, deact (ms) of GluAs when co-transfected with/without γ-2 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp3-v1.docx
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Supplementary file 4
Summary of p values for comparison of τw, deact.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp4-v1.docx
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Supplementary file 5
Summary of τw, des (ms) of GluAs when co-transfected with/without γ-2 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp5-v1.docx
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Supplementary file 6
Summary of p values for comparison of τw, des.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp6-v1.docx
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Supplementary file 7
Summary of τw, rec (ms) of GluAs when co-transfected with/without γ-2 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp7-v1.docx
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Supplementary file 8
Summary of p values for comparison of τw, rec.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp8-v1.docx
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Supplementary file 9
Summary of GluA1i/GluA2(R)i-G receptors when co-transfected with/without γ-2 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp9-v1.docx
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Supplementary file 10
Summary of p values for comparison of GluA1i/GluA2(R)i-G receptors when co-transfected with/without γ-2 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp10-v1.docx
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Supplementary file 11
Summary of GluA2(R)i-G/GluA3(R)i receptors when co-transfected with/without γ-2 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp11-v1.docx
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Supplementary file 12
Summary of p values for comparison of GluA2(R)i-G/GluA3(R)i receptors when co-transfected with/without γ-2 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp12-v1.docx
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Supplementary file 13
Summary of wild-type primary hippocampal neurons and ABHD6-knockout primary hippocampal neurons.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp13-v1.docx
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Supplementary file 14
Summary of p values for comparison of wild-type primary hippocampal neuron and ABHD6-knockout primary hippocampal neuron.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp14-v1.docx
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Supplementary file 15
Summary of GluA4i receptors when co-transfected with/without γ-2 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp15-v1.docx
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Supplementary file 16
Summary of p values for comparison of GluA4i receptors when co-transfected with/without γ-2 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp16-v1.docx
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Supplementary file 17
Summary of GluA1i receptors when co-transfected with/without γ-8 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp17-v1.docx
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Supplementary file 18
Summary of p values for comparison of GluA1i receptors when co-transfected with/without γ-8 and/or ABHD6.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp18-v1.docx
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Supplementary file 19
Mycoplasma test report.
- https://cdn.elifesciences.org/articles/99623/elife-99623-supp19-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/99623/elife-99623-mdarchecklist1-v1.docx
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Source data 1
File contains the underlying processed numerical values for all graphs and plots in the main figures, figure supplements, and supplementary files, as well as the original uncropped gel/blot images and raw electrophysiological recording data used for kinetic analyses.
- https://cdn.elifesciences.org/articles/99623/elife-99623-data1-v1.xlsx
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α/β-Hydrolase domain-containing 6 (ABHD6) accelerates the desensitization and deactivation of TARP γ-2-containing AMPA receptors
eLife 13:RP99623.
https://doi.org/10.7554/eLife.99623.3
